CN101493467A - Quantitative determination method for saccharified protein - Google Patents
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Abstract
The invention provides a method for determining saccharified specific protein, using two groups of antibody pairs; one group is specific to saccharified protein and the other group is specific to total protein; the saccharified specific protein and the non-saccharified specific protein can be simultaneously determined, thus obtaining the ratio thereof by calculation. The high degree of specificity and affinity of the antibody group ensures the accuracy of tests, and shortens the test time.
Description
Technical field
The present invention relates to medical detection range, relate to a kind of quantitative determination method for saccharified protein specifically.
Background technology
One. clinical meaning:
Diabetes are endocrine metabolism diseases that one group of cause of disease and pathogenesis are not understood as yet fully, and the incidence of disease is only second to cardiovascular and cerebrovascular disease and tumour at present.In China's onset diabetes rate is 2-3%, and with every millesimal speed increment.The present clinical patient's blood sugar work that detects of extensively carrying out.But blood sugar detection is only represented blood sugar level at once, and prompting patient health at that time can not be as the index of estimating the disease control degree.
Diabetes regular meeting causes pathology and various complication such as view, kidney, nerve.Will thoroughly cure and be unusual difficulty, strictly glucose level control is treatment diabetes and the effective means of preventing its various complication.The glycated proteins index is subjected to clinical great attention just day by day in recent years, and wherein, glycosylated hemoglobin and glycosylated albumin are even more important.Glycosylated albumin, glycosylated hemoglobin and glycated protein are the same, are taken as the index that is used for glycemic control.The glycosylated hemoglobin reflection is 1 month～2 months blood sugar level in the past, and the glycosylated albumin and the blood sugar level in glycated protein reflection 2 week～3 weeks of past.The detection of glycosylated albumin has avoided seralbumin to descend to the influence of measurement result.Therefore when understanding the blood sugar short-term level, has advantage during as gestation, wound, acute complication.
That a part of haemoglobin that glycosylated hemoglobin is meant in the blood and glucose combines.When concentration of glucose was higher in the blood, the formed saccharification hemoglobin content of human body also can be higher relatively.The erythrocytic life-span was generally 120 days in the human body, and before cell death, saccharification hemoglobin content also can keep constant relatively in the blood.Because of a little glycosylated hemoglobin levels reflections be average blood sugar level in preceding 120 days of detection, and with the blood drawing time, on an empty stomach whether whether patient use factor such as insulin irrelevant.It is the good index of judging the diabetes long-term control.
Albumin is the highest protein of content in the blood.Glycosylated albumin is formed at the reaction between blood sugar and the albumin.Reaction is slowly and the rising of the glycosylated albumin that continues means the lasting rising of blood sugar for the first period.Because albumin about fortnight cycling time in blood is measured preceding 10-20 days monitoring window so glycosylated albumin provides.
The measurement result of glycosylated hemoglobin and glycosylated albumin all is to represent with percent, refers to the ratio that accounts for whole haemoglobins (albumin) with the haemoglobin (albumin) of glucose combination.
The level of ND's glycosylated hemoglobin is 4-6%; Many diabetics of discovering if can be reduced to the glycosylated hemoglobin level below 8%, the complication of diabetes will reduce greatly, if glycosylated hemoglobin＞9%, the continued property of patient hyperglycaemia is described, nephrosis can take place, artery sclerosis, complication such as cataract, and acute complication such as ketoacidosis might appear.Therefore, relevant expert's suggestion, if diabetic's glycemic control standard up to standard, and the glycemic control state should be accepted 2 glycosylated hemoglobins every year at least and detect for steadily; Need change the data scheme for those, the perhaps patient of glycemic control state labile, and the patient who is carrying out insulinize should carry out glycosylated hemoglobin mensuration one time by every three months.
The level of ND's glycosylated albumin is 12-17%.But the assay method difference has different result, the level of reporting the glycosylated albumin of pointing out the normal person is also arranged between 1.5%-15.0%.The result of glycosylated albumin can be used for the screening of diabetes, and diabetic's glycosylated albumin level is apparently higher than the normal control group, and is proportionate with fasting blood-glucose.The mensuration of glycosylated albumin is not subjected to feed, cholerythrin, uric acid, creatinine, haemoglobin and ascorbic interference, especially got rid of liver and kidney disease, hemolytic anemia and protein structure unusually to the influence of plasma proteins amount.Glycosylated albumin changes early than glycosylated hemoglobin, and to unsettled change of blood sugar, glycosylated albumin can in time be monitored.Glycosylated albumin can be used as the discriminating of diabetes gestation and pregnancy period hyperglycaemia, and diabetes gestation person glycosylated albumin is higher than the level of pregnancy period hyperglycemic patients.Glycosylated albumin and glycosylated hemoglobin have correlativity preferably, but glycosylated hemoglobin other diseases except that diabetes also can raise, and as uremia, hyperthyroidism, renal failure etc., but glycosylated albumin is normal during these diseases; When hemolytic anemia, haemoglobin mutation body such as HbS or HbC, high HbF mass formed by blood stasis, glycosylated albumin is interference-free owing to the glycosylated hemoglobin that descends red blood cell life span changes greatly; Therefore glycosylated albumin combines with glycosylated hemoglobin to use and improves the accuracy that detects.When protein concentration changed, to nephrotic syndrome, cirrhosis, paraproteinemia or the patient after phase reaction when acute, the glycated protein result was unreliable when sick, and the influence that the mensuration of glycosylated albumin is not changed by albumen.Albumen can form the whole not product (AGE) of stable and irreversible terminal glycosylation after saccharification, thereby the 26S Proteasome Structure and Function of these protein is changed, and influences the affinity of many parts; Low-density lipoprotein (LDL) saccharification becomes LDL-AGE, immune response through body, form the LDL-AGE immune complex, promote macrophage gathering, engulf and gather cholesterol ester, make it change into foam cells, through acceptor mediation, activate, synthetic, discharge and have extensive bioactive cell factor, finally make endangium damage, atheromatous plaque form atherosclerotic.After VI ATHEROCOLLAGEN GE forms in the body simultaneously, make the crosslinked increase of covalence key in the molecule, helical structure reduces, cause collagen flexibility decrease and hardening, on the blood capillary basilar memebrane, form deposition and cause the counterdie thickening, and the autoimmune response of IgG immune complex has increased the weight of tissue damage, causes retina and glomerulus to produce microangiopathy.Therefore glycosylated albumin is measured the glycosylation that can estimate histone again, understands prediction, the prevention of generation, development and the complication of its complication.
Two. the related assays method:
The method that glycated proteins detects is a lot, and assay method mainly contains: chromatography, electrophoresis, chemical method and immunization.
At present, be the high performance liquid chromatography (HPLC) of principle with the ion-exchange chromatography, promptly the glycated protein analyser has been widely used in measuring glycated protein % in the blood.Though this method repeatability is very good, also exists some shortcomings, comprising special high performance liquid chromatograph system of needs, measure finite capacity, and the inaccurate result who introduces by multi-form haemoglobin.In addition, it needs the technical maintenance of height.
US patent #4,269,605 U.S patent #5,284,777, told about the affinity chromatography method of using a kind of boronic acid derivatives to be fixed in the agarose pearl.
US patent #5,110,745 have introduced the boronic acid compounds reaction that will measure sample and excess quantity, and then adsorb remaining boronic acid compounds with the glycosylated molecule that solidifies.Measure the amount that does not have in conjunction with the glycosylated molecule of boronic acid compounds at last.The method operation steps is a lot, also must the strict amount of controlling boronic acid compounds.
Jap.P. #6,058,936 and European patent #455225 told about a boronic acid derivatives and a detectable label coupling (as fluorescent chemicals, chemiluminescence is compound, isotope, enzyme or other labels) measured glycated proteins with another antibody then.
All said methods all are to measure single glycated proteins, and glycated proteins is normally recently to express with the percentage of total protein quality.So also must measure the total protein quality with another kind of method.
WO#9840750 has recommended a kind of method boric acid of curing and the glycated protein qualitative response in the sample.His design can be measured non-glycated proteins and obtain the result of the number percent of glycated proteins and total protein quality.But because the affinity of boric acid and glycated proteins is limited, the result of the method can be subjected to the influence of temperature and incubation time.
In recent years, immunoreagent is because the special vitality that it has shown it to the high degree of specificity and the affinity of glycated proteins.
European patent #201187 has told about the anti-glycosylated hemoglobin monoclonal antibody method of preparation specificity.
US patent #522392 has told about the anti-glycosylated albumin monoclonal antibody method of preparation specificity.
US patent #5470759 has told about and has utilized the anti-glycosylated hemoglobin monoclonal antibody of specificity, sets up immune agglutination test and immune turbidity test and measures glycosylated hemoglobin.
US patent #5206144 has told about the preparation anti-glycosylated hemoglobin monoclonal antibody method of specificity and has measured glycosylated hemoglobin with the enzyme immunity test that this antibody is set up.The method must be got the sample red blood cell only, is coated on the microwell plate after the haemolysis dilution.Then with the anti-glycosylated hemoglobin monoclonal antibody reactive of specificity after, measure glycosylated hemoglobin with enzyme mark second antibody again.The method step is a lot.
US patent #5932480 has introduced the method for single stage method mensuration glycosylated hemoglobin number percent.Concrete steps are: sample, and the solid phase of not wrapping quilt, specific antibody enzyme conjugate was hatched 40 minutes together simultaneously, 37 degree Celsius.After cleaning, add substrate again and measure enzymatic activity, result and standard items relatively obtain the number percent of sample glycosylated hemoglobin.The shortcoming of the method is: in the process that the solid phase of specific antibody enzyme conjugate and bag quilt is hatched simultaneously, background is certain to raise.
Because the content of hemoglobin in the blood is very high, much measure the immunization method of glycosylated hemoglobin, comprise above-mentioned two kinds, all wrapping by the part of haemoglobin as its determination step.For making the haemoglobin bag, usually also must add a degenerative treatments step by better effects if.Its result is often: minute prolongs, and error at measurment increases.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of quantitative determination method for saccharified protein, to solve defective of the prior art.
Quantitative determination method for saccharified protein provided by the present invention, concrete comprises the steps:
1. A-B combination: draw 5-50 microlitre haemolysis dilution in the solid phase of antibody A bag quilt, add the antibody B of 100 microlitre sample diluents, 100 microlitres detection label then, left standstill after fully shaking up room temperature 5-30 minute;
2. A-C combination: draw 5-50 microlitre haemolysis dilution in the solid phase of another antibody A bag quilt, add the antibody C of 100 microlitre sample diluents, 100 microlitres detection label then, left standstill after fully shaking up room temperature 5-30 minute;
3. A-B standard items combination: the standard items of drawing 5-50 microlitre glycated proteins and non-glycated proteins respectively are in the solid phase of different antibody A bag quilts, add the antibody B of 100 microlitre sample diluents, 100 microlitres detection label then respectively, left standstill after fully shaking up room temperature 5-30 minute;
4. A-C standard items combination: the standard items of drawing 5-50 microlitre glycated proteins and non-glycated proteins respectively are in the solid phase of different antibody A bag quilts, add the antibody C of 100 microlitre sample diluents, 100 microlitres detection label then respectively, left standstill after fully shaking up room temperature 5-30 minute;
5. the reactant liquor in all solid phases of inclining is washed five times with cleansing solution 300 microlitres then; Pat dry residual old drop;
6. in each solid phase micropore, add 100 microlitre marker enzyme substrates, left standstill room temperature 5-30 minute;
7. in each solid phase micropore, add 100 microlitres and end liquid, fully shake up back reading on readout instrument;
8. the sample data that obtain in the A-B combination compare with the combination of A-B standard items, calculate glycated proteins concentration;
9. the sample data that obtain in the A-C combination compare with the combination of A-C standard items, calculate total protein concentration;
10. glycated proteins concentration is promptly obtained the glycated proteins percent concentration divided by total protein concentration;
Wherein said glycated proteins is glycosylated albumin or glycosylated hemoglobin;
Wherein antibody A is special to gross protein; Antibody B is special to glycated proteins; Antibody C is special to gross protein.
Antibody A-glycated proteins-antibody B can form sandwich immune complex.
Specific antibody A-gross protein-antibody C can form sandwich immune complex.
The solid phase of wherein said antibody A bag quilt is common microwell plate, luminous microwell plate, fluorescence microwell plate, common microparticle or magnetic particle.
The wherein said detection label that is used for labelled antibody B, C is marker enzyme, isotope, fluorescent chemicals or chemiluminescence compound; Preferably hydrogen peroxidase, alkaline phosphatase or galactosidase.
Wherein the marker enzyme substrate of step described in 6. is 3,3 ' 5,5-tetramethyl benzidines and superoxol, chemical luminous substrate solution or fluorogenic substrate solution; Wherein preferably 3,3 ' 5,5-tetramethyl benzidines and superoxol (TMB solution).When the marker enzyme substrate was chemical luminous substrate solution or fluorogenic substrate solution, step did not need to add termination liquid in 7..
Wherein the readout instrument of step described in 7. is elisa reading instrument, chemiluminescence readings instrument, fluorescence readout instrument or isotope calculating instrument
Antibody A, B, C are the commercially available prod,
The antibody A, B, the C that wherein are used to detect glycosylated hemoglobin come from U.S. Abcam company;
Antibody A: mouse-anti human hemoglobin monoclonal antibody model: AB401
Antibody B: mouse-anti people glycosylated hemoglobin monoclonal antibody model: AB47149
Antibody C: mouse-anti human hemoglobin monoclonal antibody model: AB402
Also can be antibody with other sources of identical characteristics.
Wherein be used to detect sero-abluminous antibody A, B, C come from U.S. Abcam company and U.S. USBiologicai company;
Antibody A: U.S. Abcam company, title: goat-anti human serum albumins polyclonal antibody, affinity chromatography is pure, model: AB19180
Antibody B: U.S. USBiologicai company, title: mouse-anti human serum glycosylated albumin monoclonal antibody, model: A1327-55
Antibody C: U.S. Abcam company, title: mouse-anti human serum albumins monoclonal antibody, model: AB7793
Also can be antibody with other sources of identical characteristics.
The standard items source of glycated proteins and non-glycated proteins: USBiological company.
Wherein the haemolysis sample is prepared as follows: take 1-2 microlitre capillary whole blood, be added in 10 milliliters of haemolysis damping fluids, left standstill room temperature 20 minutes after fully shaking up.
The preparation of haemolysis damping fluid: 0.1M succinic acid damping fluid (pH 5.0) contains 0.1%Triton X-100, and 0.05% nitrine is received.
Wherein sample diluent is: 20mM TRIS-HCl damping fluid, pH 7.20.
Wherein cleansing solution is: 10mM PBS, pH 7.40,0.05% Tween-20.
TMB solution wherein: 3,3 ' 5,5-tetramethyl benzidines and superoxol (Sigma-Aldrich, T0440)
Wherein ending liquid is: 2N HCl solution.
The source of all chemicals all is a Sigma-Aldrich company.
Assay method of the present invention has following several characteristics:
1. can use Same Way, glycated proteins of quantitative measurement simultaneously and corresponding gross protein.The glycated proteins number percent of calculating thus is more objective, accurately.Can avoid by distinct methods different instruments, varying environment, different operating personnel and the error brought is disturbed.
2. method adopts two groups of antibody antithetical phrases, and one group special to glycated proteins, and another group is special to gross protein.The high special of antibody group has guaranteed the accuracy of test, and the high-affinity of antibody group can shorten test period.
3. because measurement result is to express with the ratio of glycated proteins and corresponding gross protein, so within the specific limits, sampling what can not influence measurement result.
4. invention can be used for various modes, as traditional enzyme linked immune assay, and chemiluminescence immunoassay test, fluroimmunoassay, magnetic particle immunity test etc.
5. the present invention also can be used for immune turbidity test, prepares two kinds of different antibody microballoons, and a pair of glycated proteins is special, and a pair of gross protein is special.Test respectively, the glycated proteins percentage result of calculating then.
6. test can be adopted capillary blood, and convenient sampling reduces patient's misery.
7. method of the present invention glycated proteins of quantitative measurement simultaneously and corresponding gross protein need not wrap by haemoglobin, need not make degenerative treatments, and be easy fast, accurately.
Description of drawings
Fig. 1, antibody A bag are by solid phase
The antibody B of Fig. 2, marker enzyme mark
The antibody C of Fig. 3, marker enzyme mark
Fig. 4, marker enzyme substrate
Fig. 5, A-B combination
Fig. 6, A-C combination
The standard items curve of Fig. 7, haemoglobin, wherein X-axis is a hemoglobin concentration; Y-axis is an absorbance
The standard items curve of Fig. 8, glycosylated hemoglobin, wherein X-axis is a glycosylated hemoglobin concentration; Y-axis is an absorbance
Fig. 9, albuminous standard items curve, wherein X-axis is an albumin concentration; Y-axis is an absorbance
The standard items curve of Figure 10, glycosylated albumin, wherein X-axis is a glycosylated albumin concentration; Y-axis is an absorbance
Embodiment
The following example only is used to illustrate the present invention, rather than restriction the present invention.
Embodiment 1, the mensuration of glycosylated hemoglobin.
The preparation of insolubilized antibody:
Antibody A, mouse-anti human hemoglobin monoclonal antibody, U.S. Abcam company, model: the AB401 antibody A need be coated on microwell plate (Sigma-Aldrich, M0156) on, antibody concentration, to between 10.0 mcg/ml, bag is cushioned liquid, 0.1M carbonic acid sodium bicarbonate buffer liquid in 1.0 mcg/ml, pH 9.6. package amount: 200 microlitres/micropore, ambient temperature overnight.(the room temperature sealing is spent the night for Sigma-Aldrich, C9483) 300 microlitres/micropore with the sealing damping fluid then.After the drying, kept dry is standby.
The preparation of antibody-marker enzyme conjugate
Antibody B, mouse-anti people glycosylated hemoglobin monoclonal antibody, U.S. Abcam company, model: AB47149, antibody C, mouse-anti human hemoglobin monoclonal antibody, U.S. Abcam company, model: AB402
(Sigma-Aldrich P8415) puts together with horseradish peroxidase respectively for antibody B and antibody C.Become antibody-marker enzyme conjugate.Conjugation methods is summarized as follows according to United States Patent (USP) 4,256,833: horseradish peroxidase is dissolved in 0.1M sodium carbonate-sodium bicarbonate buffer liquid, every milliliter of 8.5,5 milligrams of enzyme of pH value.Drop by drop add 1% phenyl isothiocyanate (volume/volume absolute ethyl alcohol), at room temperature simultaneously, constantly stir enzyme solutions lightly, up to the appearance that slight muddiness is arranged.At room temperature stirred lightly 2 hours.Then, drop by drop add 0.06M sodium periodate solution until the about 0.03M of final concentration.At this moment, the o-dihydroxy of the oxygenant in the carbohydrate content of enzyme is rolled into a ball the free aldehyde radical of oxidized generation.This group can with free amino group in the antibody at 0.1M sodium carbonate-sodium bicarbonate buffer liquid, under pH value 9.5 environment, at room temperature stir lightly and formed the Xi Fushi group in 1 hour, it is stable (usually to add sodium borohydride reduction then, 5 milligrams of horseradish peroxidases and 7.5 milligrams of antibody, approximately need 0.8 milligram of sodium borohydride), being further purified unreacted antibody of removal and enzyme promptly becomes antibody-marker enzyme conjugate finished product.Antibody-marker enzyme conjugate finished product can be used BioStab enzyme stabilizers (Sigma-Aldrich, 95576) to be diluted to conjugate as required and use liquid.
The preparation of haemoglobin and glycosylated hemoglobin standard items:
(USBiological company H1850-18) is diluted to a series of concentration with a biological molecule storage liquid (Sigma-Aldrich, 92889): 0,20,100,200 mg/ml to haemoglobin.And be divided in the ampoule standby.Draw before the test in 1 microlitre to the 10 milliliter haemolysis damping fluid, fully mixing.(between 160 mg/ml, the series standard product of this haemoglobin have been contained this neighborhood to the content of normal person's whole blood haemoglobin in 110 mg/ml).
(USBiological company H1850-15), is diluted to a series of concentration with a biological molecule storage liquid (Sigma-Aldrich, 92889): 0,2,20,50 mg/ml to glycosylated hemoglobin.And be divided in the ampoule standby.Draw before the test in 1 microlitre to the 10 milliliter haemolysis damping fluid, fully mixing.
The preparation of haemolysis damping fluid: 0.1M succinic acid damping fluid (pH 5.0) contains 0.1%Triton X-100,4mM EDTA, and 0.05% nitrine is received.
The preparation of sample diluent: 20mM TRIS-HCl damping fluid, pH 7.20.
The preparation of blood specimen: take 1 microlitre capillary whole blood, be added in 10 milliliters of haemolysis damping fluids, left standstill room temperature 20 minutes after fully shaking up.
Test is carried out with following method:
1. be the micropore of 4 antibody A bag quilts of series standard product preparation of haemoglobin.The standard items of drawing 20 microlitre variable concentrations respectively are in the micropore of each antibody A bag quilt.The antibody C conjugate that adds 100 microlitre sample diluents, 100 microlitre peroxidase labellings is then used liquid, leaves standstill room temperature 20 minutes after fully shaking up;
2. be the micropore of 4 antibody A bag quilts of series standard product preparation of glycosylated hemoglobin.The standard items of drawing 20 microlitre variable concentrations respectively are in the micropore of each antibody A bag quilt.Add the antibody B of 100 microlitre sample diluents, 100 microlitre peroxidase labellings then, left standstill room temperature 20 minutes after fully shaking up;
3. prepare the micropore of 2 antibody A bag quilts for each sample.Draw 20 microlitre samples in one of them micropore, add the antibody B of 100 microlitre sample diluents, 100 microlitre peroxidase labellings then, left standstill room temperature 20 minutes after fully shaking up; Draw 20 microlitre haemolysis samples in the micropore of another antibody A bag quilt, add the antibody C of 100 microlitre sample diluents, 100 microlitre peroxidase labellings then, left standstill room temperature 20 minutes after fully shaking up.
4. incline reactant liquor in all micropores is washed five times with cleansing solution 300 microlitres then; Pat dry residual old drop.
5. add 100 microlitres, 3,3 ' 5,5-tetramethyl benzidine and superoxols in each micropore, (TMB solution, Sigma-Aldrich T0440), left standstill room temperature 10 minutes.
6. in each micropore, add 100 microlitres and end liquid, fully shake up back reading on elisa reading instrument.
7. following table 1 is the result of each standard items and sample reading in the position on the microwell plate and on elisa reading instrument in the practical operation:
Table 1
Micropore A1: haemoglobin standard product 0 mg/ml, A-C combination
Micropore B1: haemoglobin standard product 20 mg/ml, A-C combination
Micropore C1: haemoglobin standard product 100 mg/ml, A-C combination
Micropore D1: haemoglobin standard product 200 mg/ml, A-C combination
Micropore E1: normal specimen N1, the A-C combination
Micropore F1: normal specimen N2, the A-C combination
Micropore G1: diabetes sample D1, A-C combination
Micropore H1: diabetes sample D2, A-C combination
Micropore A2: glycosylated hemoglobin standard items 0 mg/ml, A-B combination
Micropore B2: glycosylated hemoglobin standard items 2 mg/ml, A-B combination
Micropore C2: glycosylated hemoglobin standard items 20 mg/ml, A-B combination
Micropore D2: glycosylated hemoglobin standard items 50 mg/ml, A-B combination
Micropore E2: normal specimen N1, the A-B combination
Micropore F2: normal specimen N2, the A-B combination
Micropore G2: diabetes sample D1, A-B combination
Micropore H2: diabetes sample D2, A-B combination
Micropore A3, B3, C3, D3, reagent blank
8. the sample data that obtain in the A-C combination compare with the combination of A-C standard items, calculate total hemoglobin concentration; The sample data that obtain in the A-B combination compare with the combination of A-B standard items, calculate glycosylated hemoglobin concentration.
The result of calculation of total hemoglobin sees Table 2 (A-C combinations):
| Title | Testing position | Concentration known (mg/ml) | Reading | Calculating concentration |
| Haemoglobin standard product 1 | A1 | 0.0 | 0.067 | - |
| Haemoglobin |
B1 | 20.0 | 0.277 | - |
| Haemoglobin |
C1 | 100.0 | 1.096 | - |
| Haemoglobin standard product 4 | D1 | 200.0 | 2.063 | - |
| Normal specimen N1 | E1 | - | 1.355 | 126.2 |
| Normal specimen N2 | F1 | - | 1.291 | 119.7 |
| Diabetes sample D1 | G1 | - | 1.472 | 138.1 |
| Diabetes sample D2 | H1 | - | 1.164 | 106.8 |
Above-mentioned data are carried out conic fitting, obtain the standard items curve of Fig. 7.
The result of calculation of glycosylated hemoglobin sees Table 3 (A-B combinations):
| Title | Testing position | Concentration known (mg/ml) | Reading | Calculating concentration |
| Glycosylated hemoglobin standard items 1 | A2 | 0.0 | 0.056 | - |
| Glycosylated hemoglobin |
B2 | 2.0 | 0.120 | - |
| Glycosylated hemoglobin |
C2 | 20.0 | 0.588 | - |
| Glycosylated hemoglobin standard items 4 | D2 | 50.0 | 1.248 | - |
| Normal specimen N1 | E2 | - | 0.184 | 4.47 |
| Normal specimen N2 | F2 | - | 0.156 | 3.46 |
| Diabetes sample D1 | G2 | - | 0.531 | 17.68 |
| Diabetes sample D2 | H2 | - | 0.323 | 9.60 |
Above-mentioned data are carried out conic fitting, obtain the standard items curve of the glycosylated hemoglobin of Fig. 8.
Glycosylated hemoglobin concentration is promptly obtained the glycosylated hemoglobin percent concentration divided by total hemoglobin concentration; Embodiment 1 has tested 4 samples, wherein, and 2 normal persons, (N1, N2) 2 diabetics.(D1, D2) result of calculation sees Table 4:
The testing result of table 4 normal person and diabetes patient's glycosylated hemoglobin
| Mark this shop | Total hemoglobin concentration | Glycosylated hemoglobin concentration | The glycated proteins percent concentration |
| N1 | 126.2 | 4.47 | 3.54％ |
| N2 | 119.7 | 3.46 | 2.89％ |
| D1 | 138.1 | 17.68 | 12.8％ |
| D2 | 106.8 | 9.60 | 8.99％ |
The preparation of insolubilized antibody:
Antibody A, goat-anti human serum albumins polyclonal antibody, U.S. Abcam company, model: AB19180, antibody A be coated on microwell plate (Sigma-Aldrich, MO156) on, the preparation method is identical with embodiment 1.
The preparation of antibody-marker enzyme conjugate
Antibody B, mouse-anti human serum glycosylated albumin monoclonal antibody, U.S. USBiological company, model: A1327-55; Antibody C: mouse-anti human serum albumins monoclonal antibody, U.S. Abcam company, model: AB7793
Antibody B and antibody C need (Sigma-Aldrich P8415) puts together with horseradish peroxidase respectively.Become antibody-marker enzyme conjugate.The conjugation methods preparation method is identical with embodiment 1.
The preparation of human serum albumins and human serum glycosylated albumin standard items:
(USBiological company A1327-31) is diluted to a series of concentration with a biological molecule storage liquid (Sigma-Aldrich, 92889): 0,20,50,100 mg/ml to human serum albumins.And be divided in the ampoule standby.Draw before the test in 2 microlitres to the 10 milliliter blood plasma damping fluid, fully mixing.(between 50 mg/ml, the sero-abluminous series standard product of this person have been contained this neighborhood to the content of normal human serum albumin in 35 mg/ml).
(USBiological company A1327-53), is diluted to a series of concentration with a biological molecule storage liquid (Sigma-Aldrich, 92889): 0,2,10,50 mg/ml to the human serum glycosylated albumin.And be divided in the ampoule standby.Draw before the test in 2 microlitres to the 10 milliliter blood plasma damping fluid, fully mixing.
The preparation of blood plasma damping fluid: 0.1M phosphate buffer (pH 7.4), 4mM EDTA, 0.05% nitrine is received.
The preparation of sample diluent: 20mM TRIS-HCl damping fluid, pH 7.20.
The preparation of blood specimen: take 2 microlitre capillary whole bloods, be added in 10 milliliters of blood plasma damping fluids, fully shake up the back stand for standby use.
Test sequence, method and result: test sequence, method is identical with embodiment 1.The result is as follows:
Reading sees Table 5 in the position on the microwell plate and on elisa reading instrument for each standard items and sample:
Micropore A1: human serum albumins standard items 0 mg/ml, A-C combination
Micropore B1: human serum albumins standard items 20 mg/ml, A-C combination
Micropore C1: human serum albumins standard items 50 mg/ml, A-C combination
Micropore D1: human serum albumins standard items 100 mg/ml, A-C combination
Micropore E1: normal specimen N1, the A-C combination
Micropore F1: normal specimen N2, the A-C combination
Micropore G1: normal specimen N3, the A-C combination
Micropore H1: diabetes sample D1, A-C combination
Micropore A2: human serum glycosylated albumin standard items 0 mg/ml, A-B combination
Micropore B2: human serum glycosylated albumin standard items 2 mg/ml, A-B combination
Micropore C2: human serum glycosylated albumin standard items 10 mg/ml, A-B combination
Micropore D2: human serum glycosylated albumin standard items 50 mg/ml, A-B combination
Micropore E2: normal specimen N1, the A-B combination
Micropore F2: normal specimen N2, the A-B combination
Micropore G2: normal specimen N3, the A-B combination
Micropore H2: diabetes sample D1, A-B combination
Micropore A3, B3, C3, D3, reagent blank
The sample data that obtain in the A-C combination compare with the combination of A-C standard items, calculate total human serum albumins concentration; The sample data that obtain in the A-B combination compare with the combination of A-B standard items, calculate human serum glycosylated albumin concentration.
The result of calculation of total human serum albumins sees Table 6 (A-C combinations):
| Title | Testing position | Concentration known (mg/ml) | Reading | Calculating concentration |
| Human serum albumins standard items 1 | A1 | 0.0 | 0.092 | - |
| Human serum albumins |
B1 | 20.0 | 0.546 | - |
| Human serum albumins |
C1 | 50.0 | 1.236 | - |
| Human serum albumins standard items 4 | D1 | 100.0 | 2.476 | - |
| Normal specimen N1 | E1 | - | 1.161 | 46.71 |
| Normal specimen N2 | F1 | - | 0.987 | 39.33 |
| Normal specimen N3 | G1 | - | 1.073 | 42.99 |
| Diabetes sample D1 | H1 | - | 0.965 | 38.39 |
Above-mentioned data are carried out the standard items curve that conic fitting obtains the human serum albumins of Fig. 9.
The result of calculation of human serum glycosylated albumin sees Table 7 (A-B combinations):
| Title | Testing position | Concentration known (mg/ml) | Reading | Calculating concentration |
| Human serum glycosylated albumin standard items 1 | A2 | 0.0 | 0.074 | - |
| Human serum glycosylated albumin |
B2 | 2.0 | 0.184 | - |
| Human serum glycosylated albumin |
C2 | 20.0 | 0.516 | - |
| Human serum glycosylated albumin standard items 4 | D2 | 50.0 | 1.647 | - |
| Normal specimen N1 | E2 | - | 0.284 | 4.46 |
| Normal specimen N2 | F2 | - | 0.313 | 5.12 |
| Normal specimen N3 | G2 | - | 0.213 | 2.86 |
| Diabetes sample D1 | H2 | - | 0.523 | 10.11 |
Above-mentioned data are carried out the standard items curve that conic fitting obtains the human serum glycosylated albumin of Figure 10.
Human serum glycosylated albumin concentration is promptly obtained human serum glycosylated albumin percent concentration divided by total human serum albumins concentration; Embodiment 2 has tested 4 samples, wherein, and 3 normal persons, (N1, N2, N3) 1 diabetic (D1).Result of calculation sees Table 8:
| Mark this shop | Total human serum albumins concentration | Human serum glycosylated albumin concentration | Glycated protein albumen percent concentration |
| N1 | 46.71 | 4.46 | 9.54％ |
| N2 | 39.33 | 5.12 | 13.02％ |
| N3 | 42.99 | 2.86 | 6.66％ |
| D1 | 38.39 | 10.11 | 26.33％ |
Claims (8)
1. a quantitative determination method for saccharified protein is characterized in that this method comprises the steps:
1. A-B combination: draw 5-50 microlitre haemolysis dilution in the solid phase of antibody A bag quilt, add the antibody B of 100 microlitre sample diluents, 100 microlitres detection label then, left standstill after fully shaking up room temperature 5-30 minute;
2. A-C combination: draw 5-50 microlitre haemolysis dilution in the solid phase of another antibody A bag quilt, add the antibody C of 100 microlitre sample diluents, 100 microlitres detection label then, left standstill after fully shaking up room temperature 5-30 minute;
3. A-B standard items combination: the standard items of drawing 5-50 microlitre glycated proteins and non-glycated proteins respectively are in the solid phase of different antibody A bag quilts, add the antibody B of 100 microlitre sample diluents, 100 microlitres detection label then respectively, left standstill after fully shaking up room temperature 5-30 minute;
4. A-C standard items combination: the standard items of drawing 5-50 microlitre glycated proteins and non-glycated proteins respectively are in the solid phase of different antibody A bag quilts, add the antibody C of 100 microlitre sample diluents, 100 microlitres detection label then respectively, left standstill after fully shaking up room temperature 5-30 minute;
5. the reactant liquor in all solid phases of inclining is washed five times with cleansing solution 300 microlitres then; Pat dry residual old drop;
6. in each solid phase micropore, add 100 microlitre marker enzyme substrates, left standstill room temperature 5-30 minute;
7. in each solid phase micropore, add 100 microlitres and end liquid, fully shake up back reading on readout instrument;
8. the sample data that obtain in the A-B combination compare with the combination of A-B standard items, calculate glycated proteins concentration;
9. the sample data that obtain in the A-C combination compare with the combination of A-C standard items, calculate total protein concentration;
10. glycated proteins concentration is promptly obtained the glycated proteins percent concentration divided by total protein concentration;
Wherein said glycated proteins is glycosylated albumin or glycosylated hemoglobin;
Wherein antibody A is special to gross protein; Antibody B is special to glycated proteins; Antibody C is special to gross protein.
2. method according to claim 1, the solid phase that it is characterized in that described antibody A bag quilt are common microwell plate, luminous microwell plate, fluorescence microwell plate, common microparticle or magnetic particle.
3. method according to claim 1, the detection label that it is characterized in that described labelled antibody B, C are marker enzyme, isotope, fluorescent chemicals or chemiluminescence compound.
4. method according to claim 1 is characterized in that the marker enzyme substrate described in step 6. is 3,3 ' 5,5-tetramethyl benzidines and superoxol, chemical luminous substrate solution or fluorogenic substrate solution.
5. method according to claim 1 is characterized in that the readout instrument described in step 7. is elisa reading instrument, chemiluminescence readings instrument, fluorescence readout instrument or isotope calculating instrument.
6. method according to claim 3 is characterized in that described marker enzyme is hydrogen peroxidase, alkaline phosphatase or galactosidase.
7. method according to claim 4 is characterized in that described marker enzyme substrate is 3,3 ' 5,5-tetramethyl benzidine and superoxols.
8. method according to claim 4 when it is characterized in that described marker enzyme substrate is chemical luminous substrate solution or fluorogenic substrate solution, does not need to add termination liquid.
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